JP4182191B2 - Ultrasound imaging of tissue perfusion by contrast energy pulse energy interference - Google Patents

Abstract

A method of measuring tissue perfusion in a human or non-human animal subject which comprises administering an effective amount of an ultrasound contrast agent to said subject, irradiating tissue in a target region with at least one pulse of ultrasound having energy sufficient to destroy or discernibly modify the echogenic properties of substantially all contrast agent in said target region, and ultrasonically detecting and quantifying the rate of flow of either further contrast agent into said target region or modified contrast agent out of said target region.

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to ultrasound imaging, and more particularly to the use of ultrasound imaging to measure tissue perfusion.
Prior art Contrast agents containing a dispersion of gas microbubbles are well known to be particularly efficient backscatterers of ultrasound because the microbubbles are low density and easy to compress. It has been. Such microbubble dispersions, when properly stabilized, can allow for very efficient ultrasound visualization of, for example, vasculature and tissue microvasculature, often at advantageously low doses.
Problems to be solved by the invention The measurement of tissue perfusion involves, for example, tumor detection, typically detection of tumor tissue with blood vessels different from healthy tissue, and blood supply to the myocardium, for example. It is important for myocardial research to evaluate. Contrast agent detection methods using current ultrasound imaging techniques may provide information about whether a particular organ or region thereof has been perfused, but do not facilitate perfusion quantification. Such information is useful for assessing whether a patient is at risk due to hypoperfusion and therefore may be useful for prophylaxis and / or treatment, but now scintigraphy, positron emission Must be obtained using radioisotope imaging techniques such as tomography or single photon emission computed tomography. All such techniques involve injection of radioactive material, potentially posing a safety hazard for both patients and medical personnel, and using expensive imaging equipment. This hinders their spread.
The present invention facilitates the relative velocity of tissue perfusion in any tissue that is sensitive to ultrasound imaging, as ultrasound imaging involving ultrasound-induced destruction or modification of contrast agents can provide a measure of tissue perfusion. And based on the knowledge that it can be measured inexpensively.
Currently, there are limitations to the prior art related to ultrasound imaging including contrast agent destruction. US-A-5425366 states that certain particulate ultrasound contrast agents, such as gas-containing polymer microcapsules, may be used by the color Doppler technique, even though they are essentially less moving as a result of uptake by the reticuloendothelial tissue system. It is stated that it can be visualized. It is proposed that the relatively high amount of irradiation energy associated with the color Doppler test ruptures the microparticles and generates a Doppler-sensitive signal described as “acoustically stimulated acoustic emission”. In practice, however, the examiner is likely to interpret the backscattered signal discontinuity as a result of the movement and produces an appropriate display. It will be appreciated that this technique is inherently inapplicable to measuring the rate of perfusion, as it relates exclusively to the detection of contrast agent microparticles that are essentially stationary.
US-A-5456257 applies a coated microbubble contrast agent in a patient's body by applying ultrasonic pulsed irradiation of an amount of energy sufficient to destroy the coated microbubble and counteracts the microbubble destruction event. The detection is described by confirmation using a reactive detection method (eg, encapsulated detection method) and subsequent fractionation of echoes received from ultrasonic transmission. After the initial transmission, the acoustic energy emitted from the microbubble destruction site is received by an ultrasonic transducer, and the resulting signal waveform is subjected to amplification detection. Echoes received from subsequent transmissions are detected in a similar manner, and signals from the two reception periods are distinguished on a spatial basis. Typically, the signal derived from the second reception period is subtracted from the signal derived from the first reception period to generate a signal exiting the microbubble destruction event, excluding other signals. A threshold may be applied to remove variations resulting from tissue movement and flowing fluid. The signal is processed using a persistence system and / or by counting events within a given area of the body over time, thereby providing an indication of the bulk (net) flow of contrast-containing blood, eg into the ventricle Although not possible, it has not been suggested that this technique can be used to quantify capillary blood flow in tissues and to measure perfusion.
Means for solving the problem The present invention also applies a recognizable amount of contrast agent in the target area, rather than using subsequent pulses to detect the background signal subtracted from the first detection series. The first high energy ultrasound pulse or series of pulses is used to disrupt or alter the appreciably, and the present invention uses a “new” or unaltered contrast agent flow to the target area (and thus blood flow). Subsequent pulses are used to detect (flow). This allows the determination of parameters such as vascular blood volume fraction, average transit time and tissue perfusion for local vascular conditions within the target area. Additional contrast that is easily detectable and quantifiable by ultrasound imaging, since the initial high energy pulse can be used, for example, to reveal a well-defined target region of detectable contrast agent The sharp front of the agent then flows into this area. Due to the ability to generate a sharp front of the moving contrast agent within the target area of interest, the method is substantially advantageous over previous attempts to estimate the rate at which contrast agent flows into the tissue immediately after injection of the contrast agent. Method. This is because the injected contrast agent front is inevitably smoothed or smeared by passing through the lungs and heart. Alternatively, the initial pulse can be used to alter the echogenicity of the contrast agent, for example by activating it in the form of a precursor, causing an increase in echogenicity in the target area. The time course of echogenic changes during and after ultrasound exposure will provide information about local vascular conditions, such as local blood volume and perfusion. For example, the flow rate of contrast agent activated by ultrasound exposure can be determined and thus used to create a perfusion map.
Thus, according to one aspect of the present invention, there is provided a method for measuring tissue perfusion in a human or non-human animal subject comprising administering an effective amount of an ultrasound contrast agent to the subject, Tissue is irradiated with at least one pulse of ultrasound having sufficient energy to disrupt or distinguishably change the echogenicity of a recognizable amount of contrast agent in the target area and enters the target area Detecting and quantifying the flow rate of additional contrast agent or altered contrast agent exiting the target area with ultrasound.
Viewed from another aspect, the present invention provides the use of an ultrasound contrast agent in the manufacture of a diagnostic material for use in a method of measuring tissue perfusion in a human or non-human animal subject, said method comprising ultrasound contrast imaging. An effective amount of an agent is administered to the subject, and the target region tissue has sufficient energy to disrupt or distinguishably change the echogenicity of a recognizable amount of contrast agent in the target region. Irradiating at least one pulse of sound waves and detecting and quantifying the flow rate of additional contrast agent entering or exiting the target area with ultrasound.
According to the present invention, a wide range of ultrasound contrast agents can be used, and the most commonly used contrast agents will be gas-containing or gasogenic. Representative examples of such contrast agents include anti-aggregation surface films (eg gelatin as described in WO-A-8002365), filmogenic proteins (eg US-A-4718433, US-A-4774958). , US-A-4844882, EP-A-0359246, WO-A-9112823, WO-A-9205806, WO-A-9217213, WO-A-9406477 or WO-A-9501187, for example Albumin such as human serum albumin), polymeric materials (eg synthetic biodegradable polymers described in EP-A-0398935, elastic interfacial synthetic polymer membranes described in EP-A-0458745, described in EP-A-0441468 Fine particulate biodegradable polyaldehyde, polyamino acid-polycyclic imide fine particulate N-dicarboxylic acid derivative described in EP-A-0458079, or WO-A-9317718 or WO-A-9607434 Biodegradable polymers), non-polymers and non-polymerizable wall-forming materials (eg those described in WO-A-9521631), or surfaces Activators (eg polyoxyethylene-polyoxypropylene block copolymer surfactants such as Pluronic, polymer surfactants described in WO-A-9506518, eg WO-A-9211873, WO-A-9217212, WO -A-9222247, WO-A-9428780, WO-A-9503835, WO-A-9640275 or WO-A-9729783 film-forming surfactants such as phospholipids) For example, gas microbubbles that are at least partially enclosed) are included.
Other useful gas-containing contrast agents include gas-containing solid systems, such as those containing or bound to gas (eg EP-A-0122624, EP-A-123235, EP-A-0365467, WO- A-9221382, WO-A-9300930, WO-A-9313802, WO-A-9313808 or WO-A-9313809, for example, gas adsorbed on the surface and / or gas is voids, cavities or pores Fine particles (particularly aggregates of fine particles).
Multi-component contrast agent formulations including, for example, a dispersed gas phase-containing composition and a composition comprising a volatile component that can be transferred to the dispersed gas phase in vivo, for example, by diffusion, would also be useful. Such contrast agents are described in the specification of the applicant's unpublished international patent application No. PCT / GB97 / 02898. They can be produced in the form of precursors if desired, and the contrast agents show significant echogenic properties only after activation by high energy sonication.
The entire disclosures of the above documents relating to gas-containing contrast agents are included in this application for reference.
Phospholipid-containing compositions are used according to the present invention, for example in the form of phospholipid-stabilized gas microbubbles, but representative examples of useful phospholipids include lecithin (ie, phosphatidylcholine) such as egg yolk lecithin or soy lecithin Natural lecithins, and synthetic or semi-synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; phosphatidic acid; phosphatidylethanolamine; phosphatidylserine; phosphatidylglycerol; phosphatidylinositol; Any of the foregoing fluorinated analogs; and mixtures of any of the foregoing with other lipids such as cholesterol. Phospholipids, such as natural (eg, soy or egg yolk derived), semi-synthetic (eg, partially synthetic) (eg, partially derived), each containing predominantly (eg, at least 75%) molecules bearing a net overall charge, eg, negative charge The use of (or fully hydrogenated) and synthetic phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and / or cardiolipin will be particularly advantageous.
Any biocompatible gas may be present in the microbubbles according to the present invention. The term “gas” as used herein includes any substance (including mixtures) that is substantially or completely in the form of a gas (including vapor) at a normal human body temperature of 37 ° C. The gas is therefore for example air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon or krypton; sulfahexafluoride, disulfadecafluoride or trifluoromethylsulfapentafluoride. Sulfur fluorides such as; selenium hexafluoride; optionally hydrogenated silanes such as methylsilane or dimethylsilane; low molecular weight hydrocarbons (eg containing up to 7 carbon atoms) such as methane, ethane, propane, butane Or alkanes such as pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, alkenes such as ethylene, propene, propadiene or butene, alkynes such as acetylene or propyne; ethers such as dimethyl ether; ketones; Ether; (including, for example, up to 7 carbon atoms) a halogenated low molecular weight hydrocarbon; or mixtures of any of the included. Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms; therefore biocompatible halogenated hydrocarbon gases are, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane. Chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g. Perfluoro-n-butane optionally mixed with isomers such as perfluoro-iso-butane), perfluoropentane, perfluorohexane and perflurane Perfluoroalkanes such as loheptane; perfluoroalkenes such as perfluoropropene, perfluorobutene (eg perfluorobut-2-ene) and perfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocyclobutane, perfluoromethylcyclobutane , Perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloalkanes such as perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (eg, perfluorinated) ketones such as perfluoroacetone, and fluorinated (eg, perfluorinated) ethers such as perfluorodiethyl ether. The use of perfluorinated gases, such as sulfahexafluoride and perfluoropropanes such as perfluoropropane, perfluorobutane and perfluoropentane, is particularly advantageous because of the high stability observed in the flow of microbubbles containing such gases. Will.
The initial high-energy ultrasonic pulse may e.g. destroy the gas-containing contrast agent, e.g. by destroying a stabilizing surface film such as a protein, polymer or film-forming surfactant and / or around the gas-containing material Can be caused by promoting dissolution of tissue into tissue fluid. However, it is not necessary to completely destroy the echogenic nature of the contrast agent in the target area. It is sufficient to change the “acoustic signature” so that it can be ultrasonically distinguished from the “new” contrast agent that subsequently flows in. Thus, for example, partial or complete ultrasound-induced collapse of an encapsulating shell material generates gas microbubbles that vibrate more freely than contrast agent encapsulated gas, and have been modified to distinguish acoustic properties. Can be shown. Alternatively, the initial sonication can cause other echogenic changes to parameters such as the composition, size and / or mechanical properties of the contrast agent portion. This can be used, for example, to guide activation of a contrast agent in the form of a precursor.
If desired, the contrast agent can be designed to be particularly sensitive to disruption by the initial ultrasound pulse, thereby limiting the intensity required for the initial ultrasound irradiation. This can be achieved, for example, by using a monolayer of stabilizing amphiphilic lipid material; charged amphiphilic lipid materials, such as negatively charged phospholipids, can be electronically repelled between charged lipid membranes. As a result, the formation of a single layer can be encouraged.
A variety of ultrasound imaging techniques can be used to detect and quantify another contrast agent that flows after the initial ultrasound exposure, e.g., to determine the amount of time related contrast agent that flows into the target area. Generate perfusion related images to be displayed, thereby allowing discrimination between different perfusion regions. Desired images are obtained from individual scan line analysis or from frame analysis on a frame basis; the former is a region of high perfusion rate to obtain a sufficient number of samples to distinguish regions with different perfusions. The latter may be preferred in areas where the perfusion rate is low.
Imaging modes that may be used include B-mode and Doppler based imaging, for example pulsed wave Doppler techniques such as power Doppler imaging. Harmonics (for example, 2, 3, 4 ... times the imaging frequency), eg as described in US-A-5410516 (for example, 1/2, 1/3, 2/3 of the imaging frequency) , 3/4 ...), subharmonic (eg, 3/2, 5/4 ... times the imaging frequency), and (eg, derived from such harmonics and imaging frequency) Non-linear imaging techniques based on the effects of frequency sum or difference are optionally combined with the frequency sum produced by two incident ultrasound signals of different frequencies, as described, for example, in published US patent application Ser. No. 08 / 440,266. Alternatively, it can be used like a technique based on difference detection. The second harmonic imaging may be particularly advantageous. For example, a combination of the above techniques in second harmonic power Doppler imaging would also be useful. In general, the resulting image can be displayed, for example, as a color map representing the perfusion rate, and optionally overlaid on a conventional B-mode image of the target area.
The method of the present invention provides the energy level of the initial ultrasound pulse (and thus the size of the investigated area containing the destroyed or altered contrast agent) and the subsequent ultrasound imaging parameters, particularly the frame rate or individual pulse By appropriately selecting the time between, the desired perfusion rate can be tailored and, where appropriate, the ECG-trigger method can be used. If desired, the individual results may be spatially averaged, for example in a manner known per se.
The method also allows measurement of perfusion rates below the detection limits of conventional Doppler techniques, and is also used to estimate the relative perfusion rate in the myocardium where tissue movement renders traditional Doppler imaging ineffective. obtain. Imaging of the myocardium is advantageously performed after a sufficient amount of time has elapsed since the contrast agent was injected at its peak concentration to pass through the left ventricle, so that undesired contrast-containing left ventricular blood Is weakened so that the flow rate of the contrast agent into the myocardial tissue reaches an appropriate steady state. The essentially random flow pattern exhibited by blood and contrast agents at the capillary level is such that when the method of the present invention is used at this level, the dominant flow pattern is substantially perpendicular to the scanning system and thus relatively Avoid the possibility of anomalous results that can become undetectable. The method also has the advantage that the result is substantially independent of backscatter from the myocardium itself. This level of backscattering results in different echogenicity of various regions of the myocardium and different attenuation of adjacent tissues, fluids, etc. between such regions and the ultrasonic transducer, resulting in This is beneficial because it can vary considerably in different areas. Furthermore, the results obtained by the method are not affected by the dose of contrast agent administered, provided that the initial ultrasound exposure is sufficient to change or destroy the echogenic effects of the contrast agent. .
The following non-limiting examples illustrate the invention.
[Brief description of the drawings]
In the accompanying drawings, FIGS. 1-3 are standardized time-intensity curves respectively obtained according to the procedure of Example 1-3. Intensity is expressed in linear units (LU).
Example 1
An anesthetized dog is injected intravenously with 2 ml of an aqueous suspension of a gas-containing particulate contrast agent described in WO-A-9317718. One minute after injection, the heart is scanned for approximately 10 seconds using a Vingmed Sound System 5 scanner transmitting at 3.7 MHz and high acoustic power to destroy all contrast agent in the imaged fragment. The output is then rapidly reduced by 12 dB to obtain a 12 dB increase in compensation gain, and B-mode frames for several subsequent heart beats are scanned-converted and digitally stored. A plot of normalized signal intensity against time on a linear scale is generated as shown in FIG. Each time point represents the average of a 5 × 5 pixel area. This plot shows high power sonication starting at 5 seconds. The inflow curve begins at 15 seconds when the output power decreases and shows a half peak time of about 0.7 seconds (half time), indicating that the imaging area has been successfully perfused. The half-time image is generated by matching the inflow half-time with a monotonic exponential curve. This image representing the perfusion image is pseudo-colored and superimposed on the B-mode image. The perfusion image displays normal perfusion for all regions of the myocardium.
Example 2
The left coronary artery of an anesthetized dog was partially closed to reduce coronary blood flow to a region of the myocardium, thereby causing symptoms of coronary artery stenosis. The dog was then intravenously injected with 2 ml of an aqueous suspension of gas-containing particulate contrast agent described in WO-A-9317718. One minute after injection, the heart was scanned for about 10 seconds using an HDI 3000 scanner with a P5-3 transducer transmitting at 2.7 MHz and medium acoustic power, destroying a significant fraction of contrast in the imaged fragment. . Set the ECG-trigger mode to receive the scanner rapidly at 45.4MHz (ie second harmonic) and store the end-systolic frame for several heartbeats digitally did. As a result of the reduction in overall ultrasound exposure, minimal contrast agent disruption occurred during operation in ECG-trigger mode, so the inflow curve showing the inflow of new contrast agent is from the second harmonic signal intensity. Easily led. FIG. 2 shows a plot of normalized signal intensity against time on the resulting linear scale, where each point represents the average of a 5 × 5 pixel area. White circles show results from the hypoperfused area and black circles are taken from the normally perfused area. The zero on the time axis corresponds to the time when the ECG-trigger starts. The drawn line shows the minimum mean square fit for these two data sets. The solid line represents the curve for the normally perfused area and shows a half peak time of about 0.7 seconds. The dashed line represents the curve for the hypoperfusion region and shows a half peak time of about 6 seconds, ie about 8 times the normal time. A false colored image representing the inflow half time was created and overlaid on the B-mode image.
Example 3
(A) Hydrogenated phosphatidylserine (100 mg) in a 2% solution of propylene glycol in pure water (20 ml) is heated to 80 ° C. for 5 minutes and the resulting dispersion is allowed to cool to room temperature overnight. Transfer 1 ml portions to 2 ml gavel, flush the headspace above each piece with perfluorobutane gas, and shake the beak for 45 seconds using an Espe CapMix ™ mixer for dental materials to give a milky white micro A bubble dispersion is obtained.
(B) A sample of the milky white dispersion prepared in (a) above is washed three times by centrifugation, the supernatant is removed, and then the same amount of 10% sucrose solution is added. The resulting dispersion is lyophilized and then redispersed in distilled water to give a milky white microbubble dispersion having a volume of 3.5 μm in median diameter as measured using a Coulter counter.
(C) Hydrogenated phosphatidylserine (100 mg) in pure water (20 ml) is heated to 80 ° C. for 5 minutes and the resulting dispersion is cooled to 0 ° C. overnight. 1 ml of the dispersion is transferred to a 2 ml kettle and 200 μl of 2-methylbutane (boiling point 28 ° C.) is added to it. Shake the gavel with CapMix ™ for 45 seconds to obtain an emulsion of diffusible ingredients, which is stored at 0 ° C. when not in use.
A syringe containing an amount corresponding to 2 μl of the gas content of the perfluorobutane gas dispersion prepared in (b) above and a syringe containing an amount corresponding to 2 μl of the gas content of the 2-methylbutane emulsion prepared in (c) above Prepare and inject the contents into the dog using a Y-connector and a catheter inserted into the superior paw vein at the same time.
One minute after injection, the heart is scanned for approximately 10 seconds using an ATL HDI 3000 scanner that transmits at 2.7 MHz and high acoustic power and receives at 5.4 MHz. This high-energy ultrasonic irradiation generates semi-stable free gas microbubbles and significantly increases the intensity of backscattering from the myocardium. The output power is then rapidly reduced by 12 dB to obtain a 12 dB increase in compensation gain, and a second harmonic frame for several subsequent heart beats is digitally stored. A plot of normalized signal intensity against time on a linear scale is generated as shown in FIG. Each time point represents the average of a 5 × 5 pixel area. This plot shows high power sonication starting at 5 seconds and producing a rapid rise of backscatter. The inflow curve begins at 15 seconds when the output power decreases and shows a half-life of about 3.5 seconds, indicating that the imaging area has been perfused. An outflow half-time pseudo-colored image is created and overlaid on the second harmonic B-mode image. The area of hypoperfusion is easily identified and graded as inadequate perfusion.

Claims (13)

A ultrasound contrast agent for use in the measurement method of tissue perfusion in an animal subject, human or non-human, the measurement how comprises administering an effective amount of said ultrasound contrast agent to the subject, the target Irradiating the tissue of the region with at least one pulse of ultrasound having sufficient energy to disrupt or distinguishably change the echogenicity of a recognizable amount of contrast agent in the target region; and signals for the flow of additional contrast agent or leaving the target area changing contrast agents enter the region detected by the ultrasonic, to allow quantitation of the velocity of the flow and its, on a linear scale An ultrasound contrast agent as described above , comprising plotting the normalized intensity of the detected signal against time . The ultrasound contrast agent of claim 1, wherein the contrast agent comprises a biocompatible gas. The ultrasonic contrast agent according to claim 2, wherein the gas contains sulfur halide or perfluorocarbon. The ultrasonic contrast agent according to claim 3, wherein the perfluorocarbon comprises perfluorobutane. The ultrasound contrast agent according to any one of claims 2 to 4, wherein the gas is stabilized by an amphiphilic lipid material. The ultrasonic contrast agent according to claim 5, wherein the amphiphilic lipid material contains a film-forming lipid. The ultrasound contrast agent according to claim 6, wherein the film-forming lipid comprises a phospholipid. The ultrasound contrast agent of claim 7, wherein at least 75% of the membrane-forming lipid comprises a negatively charged phospholipid. The ultrasound contrast agent of claim 8, wherein the negatively charged phospholipid comprises at least one phosphatidylserine. The ultrasound contrast agent according to any one of claims 1 to 9, wherein the ultrasonic detection and quantification of the flow rate of the additional contrast agent entering the target area is performed using B-mode or Doppler based imaging. . 11. The ultrasonic contrast agent according to claim 10, wherein a nonlinear imaging technique is used. 12. The ultrasound contrast agent according to any one of claims 1 to 11, wherein the flow rate of the additional contrast agent entering the target area is displayed as a color map. 13. The ultrasound contrast agent of claim 12, wherein the color map is superimposed on a conventional B-mode image of the target area.

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